Clinical Hemorheology and Microcirculation 58 (2014) 97–105 DOI 10.3233/CH-141874 IOS Press
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Impact of methylene blue in addition to norepinephrine on the intestinal microcirculation in experimental septic shock Jordan Nantaisa , Tristan C. Dumbartonb , Nizam Farahb,c , Alexander Maxanb,c , Juan Zhoub,c , Samuel Minora and Christian Lehmannb,c,d,∗ a
Department of General Surgery, Dalhousie University, Halifax, Canada Department of Anesthesia, Pain Management and Perioperative Medicine, Dalhousie University, Halifax, Canada c Department of Microbiology and Immunology, Dalhousie University, Halifax, Canada d Deparment of Pharmacology, Dalhousie University, Halifax, Canada b
Abstract. Methylene blue (MB) has been used with some success as a treatment for the vasoplegia of vasopressor-refractory septic shock. The putative mechanism of action of MB is the inhibition of endothelial nitric oxide within the microvasculature and improved responsiveness to endogenous catecholamines (norepinephrine (NE)). However, to date, no study has demonstrated the microcirculatory effect of methylene blue in septic shock. The objective of this randomized, controlled, animal study was to show, in an experimentally-induced, septic shock model in rats, the effects of MB and NE on global hemodynamics and the microcirculation. Mean arterial pressure (MAP) was drastically reduced following bacterial endotoxin (lipopolysaccharide, LPS) administration in animals not receiving vasopressors. Only the combination of NE + MB restored MAP to control levels by the end of the three hour experiment. Intravital microscopy of the microcirculation was performed in the terminal ileum in order to examine functional capillary density in intestinal muscle layers and the mucosa, as well as leukocyte activation in venules (rolling, adhesion to the endothelium). Untreated LPS animals showed a significant increase in leukocyte adhesion and a decrease in capillary perfusion in the intestinal microcirculation. In groups receiving NE or NE+MB, we observed a significant decrease in leukocyte adhesion and improved functional capillary density, indicating that microvasculature function was improved. This study suggests that methylene blue may be able to improve hemodynamics while preserving microvascular function in septic shock. Keywords: Methylene blue, norepinephrine, septic shock, microcirculation, intravital imaging
1. Introduction The most common cause of death in surgical intensive care units is septic shock [18]. Shock in sepsis occurs due to the loss of muscular tone in the blood vessels, complicated by unresponsiveness to endogenous and exogenous vasoconstricting agents and a break down in the barrier function of the blood vessel walls with concomitant intravascular fluid loss and capillary leakage [2]. One key factor leading to vascular dysfunction is an inappropriately increased production of nitric oxide (NO) within the blood vessel endothelium [10, 31, 32]. NO activates the soluble isoform of the ∗
Corresponding author: Christian Lehmann, Department of Anesthesia, Pain Management and Perioperative Medicine, QE II Health Sciences Centre, 10 West Victoria, 1276 South Park Street, Halifax, NS, B3H 2Y9, Canada. Tel.: +1 902 494 1287; Fax: +1 902 473 1035; E-mail: [email protected]. 1386-0291/14/$27.50 © 2014 – IOS Press and the authors. All rights reserved
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enzyme guanylate cyclase (GC), which in turn increases production of cyclic guanosine monophosphate (cGMP). Increased levels of cGMP cause progressive vasodilation of arteriolar smooth muscle and loss of vascular tone. Importantly, the up-regulation of cGMP also results in a proportionate decrease in intracellular cyclic adenosine monophosphate, which is a downstream mediator of the vasoconstrictor NE [8, 21]. In septic shock, elevated levels of nitric oxide result in the blood vessel endothelium receiving direct vasodilator input, as well as the inability to respond to NE-mediated vasoconstriction. Consequently, patients with septic shock are not able to maintain adequate blood pressure to perfuse vital organs, and progressive organ failure and death can occur. Septic shock has a high associated mortality rate of about 50% [19], therefore, therapeutic interventions that target and mitigate hypoperfusion and inflammation in septic shock are of significant clinical interest. Methylene blue (MB) is one such therapeutic agent, which, despite more than 100 years of use in the medical field as an antibiotic, dye and even anti-Alzheimer’s treatment [27, 29], has only relatively recently been studied for its role in treating septic shock [16, 28]. Although the mechanism of MB is not completely understood, MB has been shown to selectively inhibit both an inducible form of nitric oxide synthase (iNOS) and GC within vascular smooth muscle, and thereby decrease nitric-compound dependent vasodilation [9, 11]. The effect occurs through a decrease in the production of NO and inhibition of its downstream target GC, rendering the patient less prone to inappropriate uncompensated vasodilation. Additionally, MB may further improve hemodynamics in septic shock through an increase in cardiac function as has been suggested in previous clinical trials and animal models [5, 7]. At present, MB tends to be used in settings of overwhelming sepsis, when hypotension is refractory to traditional vasopressor support, such as norepinephrine, epinephrine and vasopressin [4, 24]. While we know that hemodynamics improve with its use, the effect of MB at the level of the capillary bed, where vital exchange of oxygen and nutrients occurs, is unknown. Additionally, it is not established how the effects on the microcirculation of MB and other vasopressors such as NE, differ, and whether or not vasoconstriction of the splanchnic microcirculation is compounded to a harmful degree through their combined use. For this reason, we opted to investigate the effect of NE at the microvascular level in isolation and in combination with MB in experimental septic shock. Intravital microscopy was used to quantify several microcirculatory variables in this model. We would posit that in combination with NE, as it is used in clinical practise, MB may improve hemodynamics without further compromising microcirculatory characteristics. 2. Methods 2.1. Animals 24 Lewis rats were obtained from Charles River Laboratories International Inc. (Wilmington, MA, USA). Animals were fed using standard rat chow and water ad libitum, kept on a 12-hour light and dark cycle, and cared for according to the Canadian Council for Animal Care guidelines. Animals were housed at the Dalhousie University Faculty of Medicine animal care facility. Experimental design was approved by the Dalhousie University Committee on Laboratory Animals. 2.2. Interventions All rats were anesthetized using sodium pentobarbital (Ceva Sante Animals, Montreal, QC, Canada) at a dosage of 54.7 mg/kg, injected intraperitoneally. Depth of anesthesia was assessed by lack of response
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to toe pinch. Additional anesthesia was provided when necessary throughout the experiment through intravenous (IV) injection of sodium pentobarbital. Depth of anesthesia was assessed every 15 minutes using toe pinches. Venous and arterial access was obtained through the femoral vessels. The skin was shaved and an incision was made over the vessels. The vascular bundle was then exposed through a combination of blunt and sharp dissection. The femoral artery and vein were ligated with silk sutures and cannulated using a PE50 polyethylene cannula (Intramedic, Becton, Dickinson and Company, 07417-1880, USA) secured with silk ties. Separate infusers were used for the arterial (B. Braun Melsungen, 8713030U, Germany) and the venous (Harvard apparatus syringe infusion pump 22, 2400-003, USA) systems. Subjects were divided into groups as follows: control (n = 10), LPS only (n = 5), LPS + NE (n = 4), and LPS + NE + MB (n = 5). Following preparation, the control subjects were infused with saline through the femoral venous catheter, and subjects in all other groups were infused with 20 mg/kg LPS from E. coli serotype O26:B6 (Sigma-Aldrich, Oakville ON, Canada). This was recorded as time point 0, and each subject was then observed for 120 minutes with measurement of mean arterial pressure (MAP; as recorded through the arterial catheter), heart rate (HR), and temperature every 15 minutes. Over this same period, each femoral line was infused with 2 mL/h of normal saline maintenance fluid. Following administration of saline or LPS as above, subjects received either no further intervention in the case of the control and LPS only groups, or vasopressor treatment in the remaining groups. The NE group received IV NE at a starting concentration of 0.01 g/kg/min. This infusion was titrated in order to maintain a MAP ≥100 mmHg within the first 60 minutes if possible and then held at the last effective dose for the remainder of the experiment. The MB group received IV MB at an infusion rate of 0.3 mg/kg/hour (no bolus dose). The NE and MB group received NE at 0.01 g/kg/min titrated to attempt to reach a MAP ≥100 mmHg as before, but at the 60 minute point, MB was added to the NE infusion at a rate of 0.3 mg/kg/hour. 2.3. Intravital Microscopy (IVM) At the 90-minute time-point, the abdomen was cleaned using alcohol swabs and a laparotomy was performed. A scalpel was used to incise the skin at the midline. Following this, the linea alba was grasped using forceps and incised using scissors in order to enter the peritoneal cavity. The terminal ileum (TI) was identified and a 3-4 cm portion was gently exteriorized with care not to compromise mesenteric blood flow. The ileum was then secured to the microscopy stage and overlaid with a cover slip. A solution of normal saline was warmed using a Hotline® system (Smith Medical, Rockland, MA) and continuously washed over the surface of the ileum in order to maintain its temperature and adherence to the cover slip. The saline superfusion was continued at a rate of 5 mL/hour throughout the microscopy portion of the experiment. Ten minutes before performance of IVM, 0.05% rhodamine-6G solution (1.5 mL/kg; Sigma-Aldrich, ON, Canada) was administered through the femoral IV to stain the leukocytes. 5% albumin–fluorescein isothiocyanate conjugate (FITC-albumin; 1 mL/kg; Sigma-Aldrich, ON, Canada) was then administered intravenously in order to facilitate identification of perfused vessels. IVM was carried out by first focusing on the collecting venules (V1) and post-capillary venules (V3) in the TI. Thirty-second videos of V1 and V3 venules were captured in order to highlight leukocyte rolling and adhesion. This was repeated for five different visual fields in each case. An analogous process was then carried out for both the longitudinal and circular muscle layers in order to observe the functional capillary density (FCD) in each layer. To examine the mucosal layer for FCD, it was first necessary to make an incision in TI, perpendicular to the mesenteric vessels. A 2 cm incision was made using
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electrocautery and the mucosal layer was exposed. Mucosal villi were observed and video-recorded as per the previous capillary beds. All videos were analyzed using ImageJ software (U. S. National Institutes of Health, Bethesda, Maryland, USA). Software was calibrated using a marker recorded by the same microscope preceding completion of the experiments. Leukocyte adherence was defined as the number of leukocytes (brightly fluorescing cells visible within the venule) that did not roll or migrate over a period of 30 seconds within the experimental field. Leukocyte rolling was defined as a count of leukocytes observed to roll past a superimposed line, perpendicular to the vessel walls within the experimental field over a 30 second period. Functional capillary density was determined for visible capillaries within the experimental window for longitudinal muscle, circular muscle, and mucosal layers of the TI. Capillaries with blood flow were carefully measured by manual tracing within the software and then the total length of capillaries was divided by the total area within which they are contained, thus giving an estimate of capillary density. 2.4. Statistics Statistical analysis was completed using GraphPad Prism Version 6 (GraphPad Software, La Jolla, California, USA) as well as Microsoft Excel 2013 (Microsoft, Redmond, Washington, USA). Data was tested for normal distribution using the Kolmogorov-Smirnov test. Data analysis was completed by way of one-way analysis of variance (ANOVA) testing with a post-hoc, Bonferroni-corrected, t-test analysis. A value of p < 0.05 was considered significant. 3. Results 3.1. Microcirculatory variables Leukocyte adhesion for V1 vessels was significantly higher in the LPS group (p < 0.05), with a 3.6-fold increase observed in this group, compared to controls (Fig. 1). This difference was not observed between the control group and either of the vasopressor groups. Similarly, leukocyte rolling was increased by 4.5 times in the V1 vessels of the LPS group, compared to the control. There was no significant difference in the groups receiving vasopressors compared to the control group (Fig. 2). No statistically significant differences were observed in leukocyte adhesion or sticking within the V3 vessels between groups (data not shown). Measurement of the FCD for the circular muscle layer of the intestinal wall exhibited a significant difference between the control and LPS groups (Fig. 3), with the FCD of the LPS group showing a 1.7-fold decrease. Statistically significant differences were not seen between the control group and the vasopressor groups. Additionally, there were no significant differences between the FCD in the intestinal villi or the longitudinal muscle layer among any of the groups (data not shown). 3.2. Vital and hemodynamic values The averages of mean arterial pressures (MAP) for each group are displayed in 30-minute intervals in Table 1. Blood pressure was significantly decreased in the LPS group in comparison to control 60 minutes and beyond (p < 0.05), whereas there was no difference seen between the vasopressor groups and the control. This exact same pattern is observed in the statistically significant increase in HR during this
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Fig. 1. Leukocyte adhesion. The number of adherent leukocytes in the collecting venules (V1) is shown for the control group (n = 10), lipopolysaccharide (LPS) group (n = 5), LPS and norepinephrine (NE) (n = 4), and LPS, NE, and methylene blue (MB) (n = 5). ∗ denotes significant difference in comparison to control (P < 0.05). Data is shown in number of cells (n) per mm2 ± standard deviation for each experimental group.
Fig. 2. Leukocyte rolling. The number of rolling leukocytes in the collecting venules (V1) is shown for the control group (n = 10), LPS group (n = 5) LPS and NE group (n = 4), and LPS, NE, and MB group (n = 5). ∗ denotes significant difference in comparison to control (P < 0.05). Data is shown in number of cells (n) per minute (min) ± standard deviation for each experimental group.
same time frame (Table 2). Temperature was held constant for each group. The combination of NE and MB was more effective in restoring MAP values over a wider time range than NE in isolation (Table 1). 4. Discussion This study demonstrates that microcirculatory disturbances caused by endotoxin-induced septic shock can be reversed with a titrated infusion of norepinephrine, and that methylene blue in addition to NE
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Fig. 3. Functional capillary density (FCD). FCD of the circular muscle shown for control group (n = 10), LPS group (n = 5), LPS and NE (n = 4), and LPS, NE, and MB (n = 5). ∗ denotes significant difference in comparison to control (P < 0.05). Data is shown in length of functioning capillary (cm) per area of muscle (cm2 ) ± standard deviation for each experimental group.
Table 1 Average mean arterial blood pressure (MAP) for each group. Values are shown for each recorded time interval. 95% confidence intervals are displayed in brackets Time
Control
0 30 60 90 120
103 (±10.6) 111 (±11.6) 115 (±7.87)† 110 (±10.0)† 112 (±13.1)†
LPS 102 (±15.3) 98 (±24.8) 80 (±19.7)∗ 78 (±25.7)∗ 83 (±29.0)∗
LPS+NE
LPS+NE+MB
107 (±13.4) 117 (±30.4) 98 (±29.9) 106 (±29.5)† 107 (±16.0)
113 (±12.1) 115 (±9.06) 109 (±5.34)† 105 (±8.02)† 110 (±10.6)†
∗
denotes significant difference in comparison to control (P < 0.05), † denotes significant difference in comparison to LPS (P < 0.05).
Table 2 Average heart rates (HR) for each group. Values are shown for each recorded time interval. 95% confidence intervals are displayed in brackets Time
Control
LPS
LPS+NE
LPS+NE+MB
0 30 60 90 120
445 (±25.9) 450 (±27.7) 424 (±32.0)† 424 (±34.5)† 411 (±25.8)†
460 (±39.8) 437 (±24.8) 503 (±43.5)∗ 500 (±20.0)∗ 484 (±28.9)∗
448 (±32.8) 458 (±49.8) 505 (±66.4) 480 (±61.6) 483 (±60.0)
496 (±53.2) 467 (±17.3) 467 (±50.8) 489 (±36.7) 486 (±34.9)
∗
denotes significant difference in comparison to control (P < 0.05). † denotes significant difference in comparison to LPS (P < 0.05).
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Table 3 Average temperature recorded for each group. Values are shown for each recorded time interval. 95% confidence intervals are displayed in brackets Time
Control
LPS
LPS+NE
LPS+NE+MB
0 30 60 90 120
37.3 (±1.11) 37.0 (±0.530) 36.8 (±0.580) 36.2 (±0.604) 36.9 (±0.465)
37.3 (±0.345) 36.3 (±1.52) 36.9 (±0.668) 37.2 (±0.786) 36.9 (±0.557)
37.5 (±0.715) 36.7 (±0.580) 37.5 (±1.14) 37.0 (±0.486) 36.6 (±0.416)
37.4 (±0.811) 36.5 (±0.465) 36.7 (±0.908) 36.8 (±0.872) 36.3 (±0.530)
does not compromise microcirculatory characteristics. Further, macrohemodynamic function, worsened in the LPS group, was improved in the NE and NE + MB groups. Only animals with the addition of MB to NE treatment showed complete restoration of physiologic MAP at the end of the experiment in our model of septic shock. Leukocyte interaction with vascular endothelium represents an established marker for inflammation and endothelial function in animal models [1, 26]. We have shown that both leukocyte adherence and rolling in V1 venules were increased in the LPS sepsis group in comparison to healthy controls but this effect was reversed in the NE and NE+MB groups. One might posit that NE not only improves hemodynamics, but also decreases microvascular inflammation. Previous studies have shown that NE can decrease serum tumour necrosis factor alpha in vitro [22] and in vivo [12] in sepsis, which suggests an important immune-modulating role for NE. The addition of MB caused a trend towards increased leukocyte-endothelial interaction in these same vessels, but this did not reach statistical significance in our study. With respect to alteration of the capillary circulation in our model, our data reveal that rats challenged with LPS have decreased FCD in the circular muscle layer of the terminal ileum. The addition of NE was sufficient to reverse the effect on FCD. NE + MB was still effective at reversing the LPS effect, but showed a trend towards decreasing the FCD. Although a similar data were observed in the capillaries of the mucosa and longitudinal muscle layer, we did not observe statistically significant changes in baseline FCD in these other capillary beds. An additional point of interest emphasized by this experiment is that although the improvements in leukocyte adherence and FCD appear to be seen in all of the samples treated with NE, this vasopressor alone was not always sufficient enough to maintain a physiologic MAP. The MAP achieved with NEalone is only intermittently higher than that of the LPS group. However, the addition of MB caused normalization of the MAP in the treatment group at all time points beyond 60 minutes, without loss of favourable microvascular characteristics. These findings support the use of MB as an adjunct when NE alone is not capable of maintaining a physiologic blood pressure, as has been supported in previous human trials [4, 7, 14–16, 23, 24, 28]. One limitations in this study is the single dose regimen employed. However, the optimal dose of MB is not known. As such, we may have been under- or over-dosing the rats in this experiment. Most case series and two controlled trials in humans [15, 18, 20] seem to have been guided by previous bolus dosing for historical indications, although a single controlled trial examined the issue of dose explicitly [13]. The most cited dose in sepsis has typically been a bolus of 1–2 mg/kg IV; however, numerous infusions have also been described, with good clinical effect being seen at doses of 0.3–2 mg/kg/hour [18]. We chose to
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use a 0.3 mg/kg/hour dosing strategy in order to best mimic the low range within the clinical arena, but this may well represent an under-dosing for the rat’s physiology. Further dose-finding studies will help to resolve this issue, as well as provide information about the optimal timing of therapy. 5. Conclusions Overall, the microvascular variables examined herein did not deteriorate by the addition of a second vasopressor therapy, i.e. MB to the standard vasopressor treatment using NE. The data also suggest that the combination of NE with MB is more effective than NE alone in the maintenance of MAP. Therefore, the practise of using MB to treat patients with septic shock unresponsive to increasing doses of NE is supported by our findings. Whether or not the utilization of MB in this setting is capable of influencing the mortality and morbidity of septic shock, and what dose of MB is most effective in this setting, remains to be determined. References [1] O. Barreiroa and F. S´anchez-Madrid, Molecular basis of leukocyte-endothelium interactions during the inflammatory response, Revista Espa˜nola de Cardiologia 62 (2009), 552-562. [2] J. Boisrame-Helms, H. Kremer, V. Schini-Kerth and F. Meziani, Endovascular dysfunction in sepsis, Curr Vasc Pharm 13 (2011), 150–160. [3] R.C. Bone, R.A. Balk, F.B. Cerra, R.P. Dellinger, A.M. Fein, W.A. Knaus, R.M. Schein and W.J. Sibbald, Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. The ACCP/SCCM Consensus Conference Committee. American College of Chest Physicians/Society of Critical Care Medicine, Chest 101 (1992), 1644–1655. [4] G. Brown, D. Frankl and T. Phang, Continuous infusion of methylene blue for septic shock, Postgraduate Medical Journal 72 (1996), 612–624. [5] A. Donati, G. Conti, S. Loggi, C. M¨unch, R. Coltrinari, P. Pelaia, P. Pietropaoli and J. Preiser, Does methylene blue administration to septic shock patients affect vascular permeability and blood volume? Critical Care Medicine 30 (2002), 2271–2277. [6] T. Dumbarton, C. Yeung, F. White, S. Minor and R. Green, Prolonged methylene blue infusion in refractory septic shock: A case report, Can J Anesth 58 (2011), 401–405. [7] O.V. Evgenov, B. Sveinbjørnsson and L.J. Bjertnaes, Continuously infused methylene blue modulates the early cardiopulmonary response to endotoxin in awake sheep, Acta anaesthesiologica Scandinavica 45 (2001), 1246–1254. [8] P.R. Evora, P.J. Ribeiro, W.V. Vicente, C.L. Reis, A.J. Rodrigues, A.C. Menardi, L. Alves, P.M. Evora and S. Bassetto, Methylene blue for vasoplegic syndrome treatment in heart surgery: Fifteen years of questions, answers, doubts and certainties, Revista Brasileira de Cirurgia Cardiovascular 24 (2009), 279–288. [9] D. Fernandes and J. Assreuy, Nitric oxide and vascular reactivity in sepsis, Shock 30 (2008), 10–13. [10] S. Forconi and T. Gori, Endothelium and hemorheology, Clin Hemorheol Microcirc 53 (2013), 3–10. [11] C.A. Gruetter, P.J. Kadowitz and L.J. Ignarro, Methylene blue inhibits coronary arterial relaxation and guanylate cyclase activation by nitroglycerin, sodium nitrite, and amyl nitrite, Canadian Journal of Physiology and Pharmacology 59 (1981), 150–156. [12] K.J. Hartemink and A.B.J. Groeneveld, Vasopressors and inotropes in the treatment of human septic shock: Effect on innate immunity? Inflammation 35 (2012), 206–213. [13] N. Juffermans, M. Vervloet, C. Daemen-Gubbels, J. Binnekade, M. de Jong and A. Groeneveld, A dose-finding study of methylene blue to inhibit nitric oxide actions in the hemodynamics of human septic shock, Nitric Oxide 22 (2012), 275–280. [14] J.F. Keaney, J.C. Puyana, S. Francis, J.F. Loscalzo, J.S. Stamler and J. Loscalzo, Methylene blue reverses endotoxin-induced hypotension, Circulation Research 74 (1994), 1121–1125.
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Impact of methylene blue in addition to norepinephrine on the intestinal microcirculation in experimental septic shock Jordan Nantaisa , Tristan C. Dumbartonb , Nizam Farahb,c , Alexander Maxanb,c , Juan Zhoub,c , Samuel Minora and Christian Lehmannb,c,d,∗ a
Department of General Surgery, Dalhousie University, Halifax, Canada Department of Anesthesia, Pain Management and Perioperative Medicine, Dalhousie University, Halifax, Canada c Department of Microbiology and Immunology, Dalhousie University, Halifax, Canada d Deparment of Pharmacology, Dalhousie University, Halifax, Canada b
Abstract. Methylene blue (MB) has been used with some success as a treatment for the vasoplegia of vasopressor-refractory septic shock. The putative mechanism of action of MB is the inhibition of endothelial nitric oxide within the microvasculature and improved responsiveness to endogenous catecholamines (norepinephrine (NE)). However, to date, no study has demonstrated the microcirculatory effect of methylene blue in septic shock. The objective of this randomized, controlled, animal study was to show, in an experimentally-induced, septic shock model in rats, the effects of MB and NE on global hemodynamics and the microcirculation. Mean arterial pressure (MAP) was drastically reduced following bacterial endotoxin (lipopolysaccharide, LPS) administration in animals not receiving vasopressors. Only the combination of NE + MB restored MAP to control levels by the end of the three hour experiment. Intravital microscopy of the microcirculation was performed in the terminal ileum in order to examine functional capillary density in intestinal muscle layers and the mucosa, as well as leukocyte activation in venules (rolling, adhesion to the endothelium). Untreated LPS animals showed a significant increase in leukocyte adhesion and a decrease in capillary perfusion in the intestinal microcirculation. In groups receiving NE or NE+MB, we observed a significant decrease in leukocyte adhesion and improved functional capillary density, indicating that microvasculature function was improved. This study suggests that methylene blue may be able to improve hemodynamics while preserving microvascular function in septic shock. Keywords: Methylene blue, norepinephrine, septic shock, microcirculation, intravital imaging
1. Introduction The most common cause of death in surgical intensive care units is septic shock [18]. Shock in sepsis occurs due to the loss of muscular tone in the blood vessels, complicated by unresponsiveness to endogenous and exogenous vasoconstricting agents and a break down in the barrier function of the blood vessel walls with concomitant intravascular fluid loss and capillary leakage [2]. One key factor leading to vascular dysfunction is an inappropriately increased production of nitric oxide (NO) within the blood vessel endothelium [10, 31, 32]. NO activates the soluble isoform of the ∗
Corresponding author: Christian Lehmann, Department of Anesthesia, Pain Management and Perioperative Medicine, QE II Health Sciences Centre, 10 West Victoria, 1276 South Park Street, Halifax, NS, B3H 2Y9, Canada. Tel.: +1 902 494 1287; Fax: +1 902 473 1035; E-mail: [email protected]. 1386-0291/14/$27.50 © 2014 – IOS Press and the authors. All rights reserved
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enzyme guanylate cyclase (GC), which in turn increases production of cyclic guanosine monophosphate (cGMP). Increased levels of cGMP cause progressive vasodilation of arteriolar smooth muscle and loss of vascular tone. Importantly, the up-regulation of cGMP also results in a proportionate decrease in intracellular cyclic adenosine monophosphate, which is a downstream mediator of the vasoconstrictor NE [8, 21]. In septic shock, elevated levels of nitric oxide result in the blood vessel endothelium receiving direct vasodilator input, as well as the inability to respond to NE-mediated vasoconstriction. Consequently, patients with septic shock are not able to maintain adequate blood pressure to perfuse vital organs, and progressive organ failure and death can occur. Septic shock has a high associated mortality rate of about 50% [19], therefore, therapeutic interventions that target and mitigate hypoperfusion and inflammation in septic shock are of significant clinical interest. Methylene blue (MB) is one such therapeutic agent, which, despite more than 100 years of use in the medical field as an antibiotic, dye and even anti-Alzheimer’s treatment [27, 29], has only relatively recently been studied for its role in treating septic shock [16, 28]. Although the mechanism of MB is not completely understood, MB has been shown to selectively inhibit both an inducible form of nitric oxide synthase (iNOS) and GC within vascular smooth muscle, and thereby decrease nitric-compound dependent vasodilation [9, 11]. The effect occurs through a decrease in the production of NO and inhibition of its downstream target GC, rendering the patient less prone to inappropriate uncompensated vasodilation. Additionally, MB may further improve hemodynamics in septic shock through an increase in cardiac function as has been suggested in previous clinical trials and animal models [5, 7]. At present, MB tends to be used in settings of overwhelming sepsis, when hypotension is refractory to traditional vasopressor support, such as norepinephrine, epinephrine and vasopressin [4, 24]. While we know that hemodynamics improve with its use, the effect of MB at the level of the capillary bed, where vital exchange of oxygen and nutrients occurs, is unknown. Additionally, it is not established how the effects on the microcirculation of MB and other vasopressors such as NE, differ, and whether or not vasoconstriction of the splanchnic microcirculation is compounded to a harmful degree through their combined use. For this reason, we opted to investigate the effect of NE at the microvascular level in isolation and in combination with MB in experimental septic shock. Intravital microscopy was used to quantify several microcirculatory variables in this model. We would posit that in combination with NE, as it is used in clinical practise, MB may improve hemodynamics without further compromising microcirculatory characteristics. 2. Methods 2.1. Animals 24 Lewis rats were obtained from Charles River Laboratories International Inc. (Wilmington, MA, USA). Animals were fed using standard rat chow and water ad libitum, kept on a 12-hour light and dark cycle, and cared for according to the Canadian Council for Animal Care guidelines. Animals were housed at the Dalhousie University Faculty of Medicine animal care facility. Experimental design was approved by the Dalhousie University Committee on Laboratory Animals. 2.2. Interventions All rats were anesthetized using sodium pentobarbital (Ceva Sante Animals, Montreal, QC, Canada) at a dosage of 54.7 mg/kg, injected intraperitoneally. Depth of anesthesia was assessed by lack of response
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to toe pinch. Additional anesthesia was provided when necessary throughout the experiment through intravenous (IV) injection of sodium pentobarbital. Depth of anesthesia was assessed every 15 minutes using toe pinches. Venous and arterial access was obtained through the femoral vessels. The skin was shaved and an incision was made over the vessels. The vascular bundle was then exposed through a combination of blunt and sharp dissection. The femoral artery and vein were ligated with silk sutures and cannulated using a PE50 polyethylene cannula (Intramedic, Becton, Dickinson and Company, 07417-1880, USA) secured with silk ties. Separate infusers were used for the arterial (B. Braun Melsungen, 8713030U, Germany) and the venous (Harvard apparatus syringe infusion pump 22, 2400-003, USA) systems. Subjects were divided into groups as follows: control (n = 10), LPS only (n = 5), LPS + NE (n = 4), and LPS + NE + MB (n = 5). Following preparation, the control subjects were infused with saline through the femoral venous catheter, and subjects in all other groups were infused with 20 mg/kg LPS from E. coli serotype O26:B6 (Sigma-Aldrich, Oakville ON, Canada). This was recorded as time point 0, and each subject was then observed for 120 minutes with measurement of mean arterial pressure (MAP; as recorded through the arterial catheter), heart rate (HR), and temperature every 15 minutes. Over this same period, each femoral line was infused with 2 mL/h of normal saline maintenance fluid. Following administration of saline or LPS as above, subjects received either no further intervention in the case of the control and LPS only groups, or vasopressor treatment in the remaining groups. The NE group received IV NE at a starting concentration of 0.01 g/kg/min. This infusion was titrated in order to maintain a MAP ≥100 mmHg within the first 60 minutes if possible and then held at the last effective dose for the remainder of the experiment. The MB group received IV MB at an infusion rate of 0.3 mg/kg/hour (no bolus dose). The NE and MB group received NE at 0.01 g/kg/min titrated to attempt to reach a MAP ≥100 mmHg as before, but at the 60 minute point, MB was added to the NE infusion at a rate of 0.3 mg/kg/hour. 2.3. Intravital Microscopy (IVM) At the 90-minute time-point, the abdomen was cleaned using alcohol swabs and a laparotomy was performed. A scalpel was used to incise the skin at the midline. Following this, the linea alba was grasped using forceps and incised using scissors in order to enter the peritoneal cavity. The terminal ileum (TI) was identified and a 3-4 cm portion was gently exteriorized with care not to compromise mesenteric blood flow. The ileum was then secured to the microscopy stage and overlaid with a cover slip. A solution of normal saline was warmed using a Hotline® system (Smith Medical, Rockland, MA) and continuously washed over the surface of the ileum in order to maintain its temperature and adherence to the cover slip. The saline superfusion was continued at a rate of 5 mL/hour throughout the microscopy portion of the experiment. Ten minutes before performance of IVM, 0.05% rhodamine-6G solution (1.5 mL/kg; Sigma-Aldrich, ON, Canada) was administered through the femoral IV to stain the leukocytes. 5% albumin–fluorescein isothiocyanate conjugate (FITC-albumin; 1 mL/kg; Sigma-Aldrich, ON, Canada) was then administered intravenously in order to facilitate identification of perfused vessels. IVM was carried out by first focusing on the collecting venules (V1) and post-capillary venules (V3) in the TI. Thirty-second videos of V1 and V3 venules were captured in order to highlight leukocyte rolling and adhesion. This was repeated for five different visual fields in each case. An analogous process was then carried out for both the longitudinal and circular muscle layers in order to observe the functional capillary density (FCD) in each layer. To examine the mucosal layer for FCD, it was first necessary to make an incision in TI, perpendicular to the mesenteric vessels. A 2 cm incision was made using
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electrocautery and the mucosal layer was exposed. Mucosal villi were observed and video-recorded as per the previous capillary beds. All videos were analyzed using ImageJ software (U. S. National Institutes of Health, Bethesda, Maryland, USA). Software was calibrated using a marker recorded by the same microscope preceding completion of the experiments. Leukocyte adherence was defined as the number of leukocytes (brightly fluorescing cells visible within the venule) that did not roll or migrate over a period of 30 seconds within the experimental field. Leukocyte rolling was defined as a count of leukocytes observed to roll past a superimposed line, perpendicular to the vessel walls within the experimental field over a 30 second period. Functional capillary density was determined for visible capillaries within the experimental window for longitudinal muscle, circular muscle, and mucosal layers of the TI. Capillaries with blood flow were carefully measured by manual tracing within the software and then the total length of capillaries was divided by the total area within which they are contained, thus giving an estimate of capillary density. 2.4. Statistics Statistical analysis was completed using GraphPad Prism Version 6 (GraphPad Software, La Jolla, California, USA) as well as Microsoft Excel 2013 (Microsoft, Redmond, Washington, USA). Data was tested for normal distribution using the Kolmogorov-Smirnov test. Data analysis was completed by way of one-way analysis of variance (ANOVA) testing with a post-hoc, Bonferroni-corrected, t-test analysis. A value of p < 0.05 was considered significant. 3. Results 3.1. Microcirculatory variables Leukocyte adhesion for V1 vessels was significantly higher in the LPS group (p < 0.05), with a 3.6-fold increase observed in this group, compared to controls (Fig. 1). This difference was not observed between the control group and either of the vasopressor groups. Similarly, leukocyte rolling was increased by 4.5 times in the V1 vessels of the LPS group, compared to the control. There was no significant difference in the groups receiving vasopressors compared to the control group (Fig. 2). No statistically significant differences were observed in leukocyte adhesion or sticking within the V3 vessels between groups (data not shown). Measurement of the FCD for the circular muscle layer of the intestinal wall exhibited a significant difference between the control and LPS groups (Fig. 3), with the FCD of the LPS group showing a 1.7-fold decrease. Statistically significant differences were not seen between the control group and the vasopressor groups. Additionally, there were no significant differences between the FCD in the intestinal villi or the longitudinal muscle layer among any of the groups (data not shown). 3.2. Vital and hemodynamic values The averages of mean arterial pressures (MAP) for each group are displayed in 30-minute intervals in Table 1. Blood pressure was significantly decreased in the LPS group in comparison to control 60 minutes and beyond (p < 0.05), whereas there was no difference seen between the vasopressor groups and the control. This exact same pattern is observed in the statistically significant increase in HR during this
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Fig. 1. Leukocyte adhesion. The number of adherent leukocytes in the collecting venules (V1) is shown for the control group (n = 10), lipopolysaccharide (LPS) group (n = 5), LPS and norepinephrine (NE) (n = 4), and LPS, NE, and methylene blue (MB) (n = 5). ∗ denotes significant difference in comparison to control (P < 0.05). Data is shown in number of cells (n) per mm2 ± standard deviation for each experimental group.
Fig. 2. Leukocyte rolling. The number of rolling leukocytes in the collecting venules (V1) is shown for the control group (n = 10), LPS group (n = 5) LPS and NE group (n = 4), and LPS, NE, and MB group (n = 5). ∗ denotes significant difference in comparison to control (P < 0.05). Data is shown in number of cells (n) per minute (min) ± standard deviation for each experimental group.
same time frame (Table 2). Temperature was held constant for each group. The combination of NE and MB was more effective in restoring MAP values over a wider time range than NE in isolation (Table 1). 4. Discussion This study demonstrates that microcirculatory disturbances caused by endotoxin-induced septic shock can be reversed with a titrated infusion of norepinephrine, and that methylene blue in addition to NE
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Fig. 3. Functional capillary density (FCD). FCD of the circular muscle shown for control group (n = 10), LPS group (n = 5), LPS and NE (n = 4), and LPS, NE, and MB (n = 5). ∗ denotes significant difference in comparison to control (P < 0.05). Data is shown in length of functioning capillary (cm) per area of muscle (cm2 ) ± standard deviation for each experimental group.
Table 1 Average mean arterial blood pressure (MAP) for each group. Values are shown for each recorded time interval. 95% confidence intervals are displayed in brackets Time
Control
0 30 60 90 120
103 (±10.6) 111 (±11.6) 115 (±7.87)† 110 (±10.0)† 112 (±13.1)†
LPS 102 (±15.3) 98 (±24.8) 80 (±19.7)∗ 78 (±25.7)∗ 83 (±29.0)∗
LPS+NE
LPS+NE+MB
107 (±13.4) 117 (±30.4) 98 (±29.9) 106 (±29.5)† 107 (±16.0)
113 (±12.1) 115 (±9.06) 109 (±5.34)† 105 (±8.02)† 110 (±10.6)†
∗
denotes significant difference in comparison to control (P < 0.05), † denotes significant difference in comparison to LPS (P < 0.05).
Table 2 Average heart rates (HR) for each group. Values are shown for each recorded time interval. 95% confidence intervals are displayed in brackets Time
Control
LPS
LPS+NE
LPS+NE+MB
0 30 60 90 120
445 (±25.9) 450 (±27.7) 424 (±32.0)† 424 (±34.5)† 411 (±25.8)†
460 (±39.8) 437 (±24.8) 503 (±43.5)∗ 500 (±20.0)∗ 484 (±28.9)∗
448 (±32.8) 458 (±49.8) 505 (±66.4) 480 (±61.6) 483 (±60.0)
496 (±53.2) 467 (±17.3) 467 (±50.8) 489 (±36.7) 486 (±34.9)
∗
denotes significant difference in comparison to control (P < 0.05). † denotes significant difference in comparison to LPS (P < 0.05).
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Table 3 Average temperature recorded for each group. Values are shown for each recorded time interval. 95% confidence intervals are displayed in brackets Time
Control
LPS
LPS+NE
LPS+NE+MB
0 30 60 90 120
37.3 (±1.11) 37.0 (±0.530) 36.8 (±0.580) 36.2 (±0.604) 36.9 (±0.465)
37.3 (±0.345) 36.3 (±1.52) 36.9 (±0.668) 37.2 (±0.786) 36.9 (±0.557)
37.5 (±0.715) 36.7 (±0.580) 37.5 (±1.14) 37.0 (±0.486) 36.6 (±0.416)
37.4 (±0.811) 36.5 (±0.465) 36.7 (±0.908) 36.8 (±0.872) 36.3 (±0.530)
does not compromise microcirculatory characteristics. Further, macrohemodynamic function, worsened in the LPS group, was improved in the NE and NE + MB groups. Only animals with the addition of MB to NE treatment showed complete restoration of physiologic MAP at the end of the experiment in our model of septic shock. Leukocyte interaction with vascular endothelium represents an established marker for inflammation and endothelial function in animal models [1, 26]. We have shown that both leukocyte adherence and rolling in V1 venules were increased in the LPS sepsis group in comparison to healthy controls but this effect was reversed in the NE and NE+MB groups. One might posit that NE not only improves hemodynamics, but also decreases microvascular inflammation. Previous studies have shown that NE can decrease serum tumour necrosis factor alpha in vitro [22] and in vivo [12] in sepsis, which suggests an important immune-modulating role for NE. The addition of MB caused a trend towards increased leukocyte-endothelial interaction in these same vessels, but this did not reach statistical significance in our study. With respect to alteration of the capillary circulation in our model, our data reveal that rats challenged with LPS have decreased FCD in the circular muscle layer of the terminal ileum. The addition of NE was sufficient to reverse the effect on FCD. NE + MB was still effective at reversing the LPS effect, but showed a trend towards decreasing the FCD. Although a similar data were observed in the capillaries of the mucosa and longitudinal muscle layer, we did not observe statistically significant changes in baseline FCD in these other capillary beds. An additional point of interest emphasized by this experiment is that although the improvements in leukocyte adherence and FCD appear to be seen in all of the samples treated with NE, this vasopressor alone was not always sufficient enough to maintain a physiologic MAP. The MAP achieved with NEalone is only intermittently higher than that of the LPS group. However, the addition of MB caused normalization of the MAP in the treatment group at all time points beyond 60 minutes, without loss of favourable microvascular characteristics. These findings support the use of MB as an adjunct when NE alone is not capable of maintaining a physiologic blood pressure, as has been supported in previous human trials [4, 7, 14–16, 23, 24, 28]. One limitations in this study is the single dose regimen employed. However, the optimal dose of MB is not known. As such, we may have been under- or over-dosing the rats in this experiment. Most case series and two controlled trials in humans [15, 18, 20] seem to have been guided by previous bolus dosing for historical indications, although a single controlled trial examined the issue of dose explicitly [13]. The most cited dose in sepsis has typically been a bolus of 1–2 mg/kg IV; however, numerous infusions have also been described, with good clinical effect being seen at doses of 0.3–2 mg/kg/hour [18]. We chose to
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use a 0.3 mg/kg/hour dosing strategy in order to best mimic the low range within the clinical arena, but this may well represent an under-dosing for the rat’s physiology. Further dose-finding studies will help to resolve this issue, as well as provide information about the optimal timing of therapy. 5. Conclusions Overall, the microvascular variables examined herein did not deteriorate by the addition of a second vasopressor therapy, i.e. MB to the standard vasopressor treatment using NE. The data also suggest that the combination of NE with MB is more effective than NE alone in the maintenance of MAP. Therefore, the practise of using MB to treat patients with septic shock unresponsive to increasing doses of NE is supported by our findings. Whether or not the utilization of MB in this setting is capable of influencing the mortality and morbidity of septic shock, and what dose of MB is most effective in this setting, remains to be determined. References [1] O. Barreiroa and F. S´anchez-Madrid, Molecular basis of leukocyte-endothelium interactions during the inflammatory response, Revista Espa˜nola de Cardiologia 62 (2009), 552-562. [2] J. Boisrame-Helms, H. Kremer, V. Schini-Kerth and F. Meziani, Endovascular dysfunction in sepsis, Curr Vasc Pharm 13 (2011), 150–160. [3] R.C. Bone, R.A. Balk, F.B. Cerra, R.P. Dellinger, A.M. Fein, W.A. Knaus, R.M. Schein and W.J. Sibbald, Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. The ACCP/SCCM Consensus Conference Committee. American College of Chest Physicians/Society of Critical Care Medicine, Chest 101 (1992), 1644–1655. [4] G. Brown, D. Frankl and T. Phang, Continuous infusion of methylene blue for septic shock, Postgraduate Medical Journal 72 (1996), 612–624. [5] A. Donati, G. Conti, S. Loggi, C. M¨unch, R. Coltrinari, P. Pelaia, P. Pietropaoli and J. Preiser, Does methylene blue administration to septic shock patients affect vascular permeability and blood volume? Critical Care Medicine 30 (2002), 2271–2277. [6] T. Dumbarton, C. Yeung, F. White, S. Minor and R. Green, Prolonged methylene blue infusion in refractory septic shock: A case report, Can J Anesth 58 (2011), 401–405. [7] O.V. Evgenov, B. Sveinbjørnsson and L.J. Bjertnaes, Continuously infused methylene blue modulates the early cardiopulmonary response to endotoxin in awake sheep, Acta anaesthesiologica Scandinavica 45 (2001), 1246–1254. [8] P.R. Evora, P.J. Ribeiro, W.V. Vicente, C.L. Reis, A.J. Rodrigues, A.C. Menardi, L. Alves, P.M. Evora and S. Bassetto, Methylene blue for vasoplegic syndrome treatment in heart surgery: Fifteen years of questions, answers, doubts and certainties, Revista Brasileira de Cirurgia Cardiovascular 24 (2009), 279–288. [9] D. Fernandes and J. Assreuy, Nitric oxide and vascular reactivity in sepsis, Shock 30 (2008), 10–13. [10] S. Forconi and T. Gori, Endothelium and hemorheology, Clin Hemorheol Microcirc 53 (2013), 3–10. [11] C.A. Gruetter, P.J. Kadowitz and L.J. Ignarro, Methylene blue inhibits coronary arterial relaxation and guanylate cyclase activation by nitroglycerin, sodium nitrite, and amyl nitrite, Canadian Journal of Physiology and Pharmacology 59 (1981), 150–156. [12] K.J. Hartemink and A.B.J. Groeneveld, Vasopressors and inotropes in the treatment of human septic shock: Effect on innate immunity? Inflammation 35 (2012), 206–213. [13] N. Juffermans, M. Vervloet, C. Daemen-Gubbels, J. Binnekade, M. de Jong and A. Groeneveld, A dose-finding study of methylene blue to inhibit nitric oxide actions in the hemodynamics of human septic shock, Nitric Oxide 22 (2012), 275–280. [14] J.F. Keaney, J.C. Puyana, S. Francis, J.F. Loscalzo, J.S. Stamler and J. Loscalzo, Methylene blue reverses endotoxin-induced hypotension, Circulation Research 74 (1994), 1121–1125.
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